Although acknowledged to be variable and subjective, manual annotation of cryo-electron tomography data is commonly used to answer structural questions and to create a “ground truth” for evaluation of automated segmentation algorithms. Validation of such annotation is lacking, but is critical for understanding the reproducibility of manual annotations. Here, we used voxel-based similarity scores for a variety of specimens, ranging in complexity and segmented by several annotators, to quantify the variation among their annotations. In addition, we have identified procedures for merging annotations to reduce variability, thereby increasing the reliability of manual annotation. Based on our analyses, we find that it is necessary to combine multiple manual annotations to increase the confidence level for answering structural questions. We also make recommendations to guide algorithm development for automated annotation of features of interest.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

The concept of atomic-scale tomography has been proposed in the past decade as a technique that could deliver the position of all atoms with high precision and their elemental (isotopic) identity. The technique was never intended to be limited to merely structural information and there is clearly a rich array of additional analytical information that can be brought to bear on such tomographs. In this paper, some of these types of information are considered and the implications are explored. The fuller realm of this analytical and structural information may be called atomic-scale analytical tomography.

Specimens that charge under electron beam irradiation in the scanning electron microscope (SEM) can be stabilized by choosing the beam energy to be such a value that the sum of the secondary and backscatter electron yields is unity, as this establishes a dynamic charge balance. We show here that for pure elements, the energies El and E2, for which charge balance occurs, are related directly to the atomic number of the material. Although generally there is no comparable relation for compounds, we also show that for polymers, the E2 energy is related both to the ratio of the number of valence electrons to molecular weight and to the electro-negativity of the monomer units that form the polymer.

Elastin-mimetic block copolymers were produced by genetic engineering. Genetically driven synthesis permitted control of the final physiochemical characteristics of the block copolymers. We designed BB and BAB block copolymers in which the A-block was hydrophilic and the B-block was hydrophobic. By designing the copolymers in this manner, it was proposed that they would self-assemble into micellar aggregates that, at high concentration, would form thermoreversible hydrogels. To analyze the three-dimensional fine surface morphology of the copolymers, to the resolution level of a few nanometers, we employed cryo-HRSEM. This method provided vast expanses of the specimen in its frozen hydrated state for survey. In our initial cryo-HRSEM studies, we observed the protein filaments and micelles surrounded by lakes of vitreous ice. Upon examination at low and intermediate magnifications, there was an extensive honeycomb-like filamentous network. To delineate the fine morphology of the hydrogel network at high magnification and to greater depths, we cryoetched away unbound water from the sample surface, in high vacuum, prior to chromium deposition. By using this technique, we were able to visualize for characterization purposes the fine fibril networks formed from the micellar aggregates over the surface of the hydrogel.

As part of an ongoing effort to increase image reproducibility and fidelity in addition to improving cross-instrument consistency, we have proposed using four separate instrument quality tests to augment the ones we have previously reported. These four tests assessed the following areas: (1) objective lens quality, (2) resolution, (3) accuracy of the wavelength information from spectral detectors, and (4) the accuracy and quality of spectral separation algorithms. Data were received from 55 laboratories located in 18 countries. The largest source of errors across all tests was user error which could be subdivided between failure to follow provided protocols and improper use of the microscope. This truly emphasizes the importance of proper rigorous training and diligence in performing confocal microscopy experiments and equipment evaluations. It should be noted that there was no discernible difference in quality between confocal microscope manufactures. These tests, as well as others previously reported, will help assess the quality of confocal microscopy equipment and will provide a means to track equipment performance over time. From 62 to 97% of the data sets sent in passed the various tests demonstrating the usefulness and appropriateness of these tests as part of a larger performance testing regiment.

Erionite samples from Rome, Oregon (USA) and Karlik, Cappadocia (Turkey) were analyzed by environmental scanning electron microscopy (E-SEM) coupled with energy-dispersive spectroscopy (EDS) to verify the chemical composition of this mineral phase, and the presence of iron in particular. By means of backscattered electron images, a large number of particles/grains were observed on the surface of the erionite fibers from both locations. The particles were found to be micrometric on samples from Rome and submicrometric on samples from Karlik, and always lighter than the hosting crystal in appearance. In different areas of the same fiber or bundle of fibers, several EDS spectra were recorded. Iron was detected only when a light particle was lying in the path of the electron beam. Iron was never identified in the EDS spectra acquired on the flat erionite surface. The results from E-SEM/EDS were confirmed by micro-Raman spectroscopy, showing bands ascribing to hematite—Fe2O3, goethite—FeO(OH), or jarosite—KFe33+(SO4)2(OH)6 when the laser beam was addressed on the light particles observed on the fiber surface. The evidence that iron is on the surface of erionite fibers, rather than being part of the crystalline structure, may be relevant for the carcinogenic potential of these fibers.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

The geometry and three-dimensional (3D) morphology of the ceria particles synthesized by spray pyrolysis (SP) from two different precursors—cerium acetate hydrate and cerium nitrate hydrate (CeA and CeN ceria particles)—were characterized by transmission electron microscopy and electron tomography. Results were compared with surface area measurements, confirming that the surface area of CeA ceria particles is twice as large as that of CeN ceria particles. This result was supported by 3D microstructural observations, which have revealed that CeA ceria particles contain open pores (connected to surfaces) and closed pores (embedded in particles), while CeN ceria particles only contained closed pores. This experimental result suggests that the type of porosity is controlled by the precursors and could be related to their melting temperature during the heating process in SP.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

2018 marked the 50th anniversary of the introduction of energy dispersive X-ray spectrometry (EDS) with semiconductor detectors to electron-excited X-ray microanalysis. Initially useful for qualitative analysis, EDS has developed into a fully quantitative analytical tool that can match wavelength dispersive spectrometry for accuracy in the determination of major (mass concentration C > 0.1) and minor (0.01 ≤ C ≤ 0.1) constituents, and useful accuracy can extend well into the trace (0.001 < C < 0.01) constituent range even when severe peak interference occurs. Accurate analysis is possible for low atomic number elements (B, C, N, O, and F), and at low beam energy, which can optimize lateral and depth spatial resolution. By recording a full EDS spectrum at each picture element of a scan, comprehensive quantitative compositional mapping can also be performed.

Two purely carbon-based functional polymer systems were investigated by bright-field conventional transmission electron microscopy (CTEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). For a carbon black (CB) filled polymer system, HAADF-STEM provides high contrast between the CB agglomerates and the polymer matrix so that details of the interface organization easily can be revealed and assignment of the CB phase is straightforward. For a second system, the functional polymer blend representing the photoactive layer of a polymer solar cell, details of its nanoscale organization could be observed that were not accessible with CTEM. By varying the camera length in HAADF-STEM imaging, the contrast can be enhanced between crystalline and amorphous compounds due to diffraction contrast so that nanoscale interconnections between domains are identified. In general, due to its incoherent imaging characteristics HAADF-STEM allows for reliable interpretation of the data obtained.

Indirect high resolution electron microscopy using one of several possible data-set geometries offers advantages over conventional high-resolution imaging in enabling the recovery of the complex wavefunction at the specimen exit plane and simultaneously eliminating the aberrations present in the objective lens. This article discusses results obtained using this method from structures formed by inorganic materials confined within the bores of carbon nanotubes. Such materials are shown to be atomically regulated due to their confinement, leading to integral layer architectures that we have termed “Feynman crystals.” These one-dimensional (1D) crystals also show a wide range of structural deviations from the bulk, including unexpected lattice distortions, and in some cases entirely new forms have been observed.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

To unambiguously evaluate the indium and nitrogen concentrations in InxGa1−xNyAs1−y, two independent sources of information must be obtained experimentally. Based on high-resolution scanning transmission electron microscopy (STEM) images taken with a high-angle annular dark-field (HAADF) detector the strain state of the InGaNAs quantum well is determined as well as its characteristic HAADF-scattering intensity. The strain state is evaluated by applying elasticity theory and the HAADF intensity is used for a comparison with multislice simulations. The combination of both allows for determination of the chemical composition where the results are in accordance with X-ray diffraction measurements, three-dimensional atom probe tomography, and further transmission electron microscopy analysis. The HAADF-STEM evaluation was used to investigate the influence of As-stabilized annealing on the InGaNAs/GaAs sample. Photoluminescence measurements show an annealing-induced blue shift of the emission wavelength. The chemical analysis precludes an elemental diffusion as origin of the energy shift—instead the results are in agreement with a model based on an annealing-induced redistribution of the atomic next-neighbor configuration.

The use of nanoparticles for the diagnosis and treatment of cancer requires the complete characterization of their toxicity, including accurately locating them within biological tissues. Owing to their size, traditional light microscopy techniques are unable to resolve them. Transmission electron microscopy provides the necessary spatial resolution to image individual nanoparticles in tissue, but is severely limited by the very small analysis volume, usually on the order of tens of cubic microns. In this work, we developed a scanning transmission electron microscopy (STEM) approach to analyze large volumes of tissue for the presence of polyethylene glycol-coated Raman-active-silica-gold-nanoparticles (PEG-R-Si-Au-NPs). This approach utilizes the simultaneous bright and dark field imaging capabilities of STEM along with careful control of the image contrast settings to readily identify PEG-R-Si-Au-NPs in mouse liver tissue without the need for additional time-consuming analytical characterization. We utilized this technique to analyze 243,000 μm3 of mouse liver tissue for the presence of PEG-R-Si-Au-NPs. Nanoparticles injected into the mice intravenously via the tail vein accumulated in the liver, whereas those injected intrarectally did not, indicating that they remain in the colon and do not pass through the colon wall into the systemic circulation.

Shear bond strength (SBS) and the interfacial adaptation (IA) of self-adhesive resin (SAR) composites to dentin were evaluated. Two SARs [Vertise Flow (VTF) and Fusio Liquid Dentin (FLD)] were evaluated and compared with a conventional restorative system [adhesive: OptiBond FL and composite: Herculite Précis (OBF/HP)]. Human third molars were used for SBS testing and IA imaging (n=7) using optical coherence tomography (OCT). Flattened dentin disks were prepared and the composites were applied into molds (2.4 mm diameter) that were positioned on dentin. Samples were subjected to SBS testing and OCT analysis, which considered an increase in signal intensity at the bonded interface as evidence of internal gaps. SBS data were analyzed by one-way analysis of variance and Tukey’s test and IA data (% distribution of high brightness values) by Kruskal–Wallis and Dunn’s test (p≤0.05). No statistically significant difference in SBS was observed between VTF (13.9±3.6 MPa) and FLD (11.3±3.2 MPa), whereas OBF/HP showed higher average strength (27.3±6.1 MPa). However, there was a statistically significant difference in IA when VTF (33.3%) was compared with FLD (1.2%) and OBF/HP (1.5%). The conventional restorative system exhibited superior SBS performance compared with SARs. However, the IA of FLD to dentin had values that were not significantly different from OBF/HP.

Abstract: The traditional methods for studying polymer microstructure in the transmission electron microscope largely hinge on the use of differential heavy-element staining to induce amplitude contrast. However, adequate staining agents are not available for all polymer systems. Furthermore, nonlinearities in the distribution of stain, particularly at interfaces, degrade the achievable resolution. Spatially resolved electron energy loss spectroscopy (EELS), on the other hand, provides a new opportunity to study polymer microstructure by providing rich spectroscopic features and high spatial resolution. Translating this opportunity into practice is underpinned by three main factors: (1) the availability of spectral fingerprints distinguishing various polymers; (2) the limits of achievable resolution; and (3) the ultimate constraints imposed by electron irradiation. This study discusses these issues and demonstrates the use of spatially resolved EELS with examples from hydrocarbon homopolymer and homopolymer blends of polyphenylene sulfide (PPS) and polyethylene terephthalate (PET), nylon 6/high-density polyethylene (HDPE) and polystyrene/polyethylene (PS/PE).

Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005

The measurement of absolute crystal polarity is crucial to understanding the structural properties of many planar defects in compound semiconductors. Grain boundaries, including twin boundaries, in the sphalerite lattice are uniquely characterized by the crystallographic misorientation of individual grains and the direction of the crystal polarity in domains adjoining the grain boundary. To evaluate crystal polarity in gallium phosphide (GaP), asymmetrical interference contrast in convergent-beam electron-diffraction (CBED) patterns was used to ascertain the nature and direction of polar bonds. The direction of the asymmetry in the electron diffraction reflections was correlated with the crystal polarity of a sample with known polarity. The CBED technique was applied to determine the polar orientation of grains adjoining Σ = 3 coherent and lateral twin boundaries in polycrystalline GaP.